Many human pathogens have developed resistance against commonly used antibiotics. This has necessitated a search for new anti-microbial substances. Besides other organisms, plants are also considered as a good source of such compounds. So far, known screening methods for testing antimicrobial activities of plants have resulted into identification of very few potential plants. To increase the chances of discovering new antimicrobials from plants, there is a need to develop alternate methods. Plants identified by the method of the present invention have shown high antimicrobial activity and tolerance to various abiotic stresses like high salt concentration, paucity of water etc. These plants can be grown in normally uncultivable lands and may be utilized as the potential source of antibiotics against selected human pathogens.
Antimicrobials have a large market share globally. Extensive screening programs are being carried out throughout the world to identify the potential plant source with new antimicrobial compounds. More than 2800 Indian plant species have so far been screened at random for different biological activities (Dhar et al. 1968, Indian J. Exp. Biol. 6:232; ibid, 1973, Indian J. Exp. Biol. 11:43; ibid, 1974, Indian J. Exp. Biol., 12:512; Bhakuni et al. 1969, Indian J. Exp. Biol. 7:250; ibid, 1971, Indian J. Exp. Biol. 9:91, ibid, 1988, Indian J. Exp. Biol., 26:883; ibid, 1990, Indian J Exp. Biol., 28:619; Dhawan et al., 1977, Indian J. Exp. Biol., 15:208; ibid, 1980, Indian J. Exp. Biol., 18:594, Aswal et al., 1984, Indian J. Exp. Biol., 22:312). However, antimicrobial activity was observed only in 45 plant species (1.6%); among these, 21 species (0.75%) exhibited antibacterial and antifungal activites. Antimicrobial activites were also observed in seeds of 35 species of Angiospems and it was found that only the seeds of 5 species (14%) had this activity. This shows that, the methods of random selection of plants for screening purposes are time consuming and costly. Besides, these offer little chance of discovering a new plant as a potential source of antibiotics (Evans, 1989, Pharmacognosy, ELBS, Bailliere Tindale, London pp. 670).
Another method used commonly for identifying plants with antimicrobial activity is through interpretation of ancient literatures and ethno-botanical data. These are merely confirmation of the activities already mentioned in these literatures. This way of identifying active plants has been moderately successful (Brantner & Grain, 1994, J. Ethnopharmacology, 44:35; Grosverhor et al., 1995, J. Ethnopharmacology, 45:97; Ieven et al., 1979, Planta Med., 36: 311; Taylor et al., 1995, J. Ethnopharmacology, 46:153; Muanza et al., 1994, Int. J. Pharmacology, 32:337; Vlietinck et al., 1995, J. Ethnopharmacology, 46:31; Weber et al., 1992, Planta Med., 58:417).
The common method currently used for testing antimicrobial activity of plant materials is as follows.
Extraction: Powdered plant materials of known quantity are exhaustively extracted with known suitable quantity of methanol either at room temperature for 2-3 weeks or using a Soxhlet extractor for 24 h. The solvent is removed at a low temperature under reduced pressure to yield a thick syrup which is suspended in methanolic water mixture (1:9) and then successively extracted with hexane, chloroform and ethyl acetate. Afforded organic solutions are dried over anhydrous sodium sulfate, filtered and concentrated to give organic extracts. Samples from methanol, hexane, chloroform and ethyl acetate extracts, as well as lyophilized aqueous fractions are further used to test for the antibacterial and antifungal activities (Ieven et al., 1979, Planta Med., 36:311).
Antimicrobial tests: Testing of the antimicrobial activity is usually performed according to the general agar plate diffusion method (Bauer et al., 1966, Am J. Clin Path. 45:493). About 10 mg of plant extract is dissolved in methanol (0.5-1 ml). Sterile blank filter paper discs of 6 mm diameter are impregnated with the resulting solutions and then asceptically deposited on the surface of innoclated plates each containing a specific test microorganism. After 48 hr of incubation at 36.degree. C. for bacteria and 72 hr of incubation at 25.degree. C. for fungi, positive results are established by the presence of clear zones of inhibition around active extracts.
Quantitative estimation of antimicrobial activity: The degree of activity is recorded in four grades according to the internal diameter (in mm) of the zones of the inhibition, incorporating the diameter of the disc (6 m): +3 (strongly active, more than 15 mm of the internal diameter), +2 (moderately active, 14-10 mm of the internal diameter), +1 (less active, internal diameter less than 9 mm) and 0 (inactive). Discs of different concentrations of common antibiotics are used as a positive control (Muanza et al., 1994, Int. J. Pharmacog., 32:337).